Sensitive and specific radioimmunoassays were used to determine the levels of cotinine, the major metabolite of nicotine catabolism, in the sera of 22 typical smokers and of two individuals who smoked cigarettes of known nicotine content under controlled conditions. In samples from the random population, cotinine was detected in concentrations ranging up to 600 ng/ml. There was poor correlation between cotinine levels and the nicotine contained in the cigarettes smoked daily by the individuals. Whether this reflects differences in smoking habits, genetic factors, and/or represents the results of enzyme induction or adaptation is unknown at present. However, since nicotine has a short plasma half-life, it may be advantageous to assay for the longer lived and more abundant metabolite in studies designed to relate the effects of smoking to health. The radioimmunoassay method for cotinine is practical for epidemiological studies and offers distinct advantages over available chromatographic techniques.